Patent Grant
8143022
The present invention relates to a high production method for infectious human hepatitis C virus particles.
Human hepatitis C virus (HCV) is a single-stranded RNA virus that causes chronic hepatitis through persistent infection. Currently, the main cause of chronic hepatitis observed worldwide is persistent HCV infection. In fact, around 50% of individuals with persistent infection develop chronic hepatitis. Chronic hepatitis in approximately 20% of these patients shifts to liver cirrhosis over the course of 10 to 20 years, and some of these patients further go on to advanced lethal pathological conditions such as hepatic cancer.
The main reasons that studies on the development of therapies for such serious diseases are hindered are the lack of efficient cellular culture systems for HCV proliferation, the lack of appropriate small animal models susceptible to HCV infection, viral replication at low levels, and genetic heterogeneity in viral genomes.
Thus, it has been expected that the development of HCV genome replication systems in cell culture systems would contribute to the understanding of viral replication and virus-cell interaction and the provision of systems for evaluation of therapeutic drugs for HCV-induced diseases.
Recently, HCV subgenomic RNA replicons have been produced as HCV-derived autonomously replicable RNAs (Patent Documents 1 and 2 and Non-Patent Documents 1-3). Thus, it becomes possible to analyze the HCV replication mechanism with the use of culture cells. Such HCV subgenomic RNA replicons are obtained by substituting structural proteins located downstream of HCV IRES in the 5′ untranslated region of HCV genomic RNA with a neomycin-resistant gene and EMCV-IRES ligated downstream thereof. By introducing such RNA replicon into a Huh7 human liver cancer cell and culturing the cell in the presence of neomycin, it was demonstrated that the RNA replicon replicates autonomously in Huh7 cells. However, only viral RNA replication, among the propagation and replication processes of HCV virus, can be evaluated in this experimental system, and thus virus particles are not produced therein. Thus, the processes of the formation of HCV virus particles in infected cells, the extracellular release of HCV particles, and infection of another cell therewith cannot be analyzed in the system.
In order to solve the above problem, a method for producing virus particles in a culture cell system has been reported. The system utilizes cDNA of HCV entire genomic RNA without the use of an RNA replicon.
Lim et al. attempted to produce HCV virus particles by treating with tetracycline a cell line obtained by introducing an expression vector in which cDNA of genomic RNA of the HCV-S1 strain (genotype 1b) is ligated downstream of a tetracycline response promoter into a Huh7 cell. They confirmed the presence of HCV particles at 1 to 6×105 copies/ml in the culture supernatant. However, they reported that such HCV particles have low infectivity (Non-Patent Document 4).
However, when HCV cDNA is expressed under the control of an RNA polymerase II-type promoter such as CMV, a CAP structure and a polyA strand are added to the 5′ end and the 3′ end of transcribed RNA, respectively. Accordingly, such RNA is used as a template for protein synthesis in a ribosome, so that replication of transcribed RNA does not take place, which is problematic.
In order to solve the above problem, Heller et al. prepared a construct that can cause intracellular synthesis of HCV RNA to which a cap and polyA are not added, by ligating a ribozyme sequence to the 5′ end and the 3′ end of the HCV genome such that it is intracellularly transcribed with RNA polymerase II and after that the transcript is cleaved with the ribozyme to produce such HCV RNA (Non-Patent Document 5). Such method for avoiding an addition of a cap at the 5′ end by means of a ribozyme is used in a method for intracellular synthesis of hairpin-type RNA (Non-Patent Document 6). In practice, it has been shown that HCV particles are produced at 1×107 copies/ml when an expression vector having an HCV construct sandwiched by two ribozymes is expressed in Huh7. Note that it has not been examined whether or not such particles exhibit infectivity.
Further, it has been recently shown that HCV particles having the ability to infect cells can be produced from HCV entire genomic RNA in a cell culture system (Patent Document 3 and Non-Patent Documents 7 and 8). In such system, the HCV particle production amount is approximately 1×107 copies/ml. Furthermore, it has been shown that it is possible to produce HCV particles having the ability to infect cells in a cell culture system with the use of chimeric viral RNA in which the non-structural protein region of the HCV con1 strain (genotype 1b) has been substituted with the gene of a viral strain (genotype 2a) (Non-Patent Document 9). No specific value for the HCV particle production amount with the use of such system has been disclosed.
Based on the above results, it has become possible to produce an experimental system that allows evaluation of the process involving the formation of HCV virus particles in infected cells, the extracellular release of HCV particles, and infection of another cell therewith.
However, the productivity of the system established by Lim et al. is low. Also, the infectivity possible with the system established by Heller et al. is unclear. Thus, it is considered that mutation might occur upon RNA replication in a system using HCV entire genomic RNA. It has been known that replication might not take place when mutation occurs in the HCV genome. In fact, it has been shown that such replication does not take place when a mutation of the GDD amino acid sequence in the NS5B protein, an HCV non-structural protein, to GND occurs. Meanwhile, the HCV particle production amount is approximately 1×107 copies/ml in both cases. Thus, further increase of the production amount has been expected.
Regarding a method for increasing HCV virus particle production amount, the production of a cell that produce a replicon at a high level has been examined. In this case, the human liver-derived Huh7 cell was used for HCV virus replication, and some cells derived from the strain were cloned. Among them, cells referred to as Huh7.5 were found to replicate approximately 3 times as many HCV RNA replicons as the parent strain (Non-Patent Document 10).
Under the above circumstances, it is thought to be important to develop a high production system for infectious HCV particles with the use of cDNA of HCV entire genomic RNA.
As a virus particle production system using cDNA corresponding to genomic RNA of an RNA virus, a system using an RNA polymerase I promoter/terminator, which is used for production of influenza virus (minus-strand RNA virus) in an animal cell system, has also been known (Non-Patent Document 11). However, it cannot be said that such influenza virus particle production system using an RNA polymerase I promoter/terminator is superior to conventional influenza virus particle production systems in terms of production amount. In addition, Non-Patent Document 11 neither describes nor suggests an HCV production system wherein HCV is a plus strand RNA virus.
It is an objective of the present invention to provide a method for high production of infectious hepatitis C virus particles from recombinant DNA in a culture cell system.
In order to establish a method for intracellularly synthesizing HCV genomic RNA having replication ability from HCV genomic cDNA, the present inventors examined HCV genomes having different genotypes and a promoter/terminator that is used for expressing the HCV genomes, and thus have found a novel combination that causes HCV genomic RNA replication. In addition, they cultured cells that synthesize such HCV genomic RNA and thereby confirmed production of infectious HCV particles at a higher level than those reported in the past. This has led to the completion of the present invention.
That is, the present invention relates to the following (a) to (c):
(a) a method for producing infectious hepatitis C virus (HCV) particles, comprising a step of introducing an expression vector into a cell that allows HCV proliferation, such expression vector comprising: JFH1 strain-derived HCV genomic cDNA downstream of a promoter recognized by a ribosomal RNA gene-derived RNA polymerase I; and DNA comprising a terminator recognized by a ribosomal RNA gene-derived RNA polymerase I, further downstream thereof;
(b) a method for producing infectious HCV particles according to (a), wherein the cell that allows HCV proliferation is selected from the group consisting of Huh7, RCYM1RC, 5-15RC, HepG2, and cells of cell lines derived therefrom; and
(c) an expression vector for producing infectious hepatitis C virus (HCV) particles, comprising: JFH1 strain-derived HCV genomic cDNA downstream of a promoter recognized by a ribosomal RNA gene-derived RNA polymerase I; and DNA comprising a terminator recognized by a ribosomal RNA gene-derived RNA polymerase I, further downstream thereof.
Further, the present invention relates to a method for producing infectious hepatitis C virus (HCV) particles, comprising the step of introducing an expression vector into a cell that allows HCV proliferation, such expression vector comprising: DNA sequences encoding the 5′ untranslated region, structural proteins, and optionally non-structural proteins of HCV and DNA sequences encoding non-structural proteins and the 3′ untranslated region derived from the HCV JFH1 strain, downstream of an RNA polymerase I promoter; and DNA comprising an RNA polymerase I terminator further downstream thereof.
More specifically, the present invention relates to the following methods of (1) to (4).
(1) A method for producing infectious hepatitis C virus (HCV) particles, comprising the step of introducing the following expression vector i) or ii) into a cell that is selected from the group consisting of Huh7, RCYM1RC, 5-15RC, HepG2, and cells of cell lines derived therefrom:
i) an expression vector comprising: DNA sequences encoding the 5′ untranslated region, Core protein, E1 protein, E2 protein, p7 protein, and NS2 protein derived from an HCV strain and DNA sequences encoding NS3, NS4A, NS4B, NS5A, and NS5B proteins and the 3′ untranslated region derived from the HCV JFH1 strain, downstream of a polymerase I promoter; and DNA comprising an RNA polymerase I terminator, further downstream thereof; or
ii) an expression vector comprising: DNA sequences encoding the 5′ untranslated region, Core protein, E1 protein, E2 protein, and p7 protein derived from an HCV strain and DNA sequences encoding NS2, NS3, NS4A, NS4B, NS5A, and NS5B proteins and the 3′ untranslated region derived from the HCV JFH1 strain, downstream of an RNA polymerase I promoter; and DNA containing an RNA polymerase I terminator, further downstream thereof.
(2) The method according to (1) above, wherein the HCV strain is a genotype 1 or genotype 2 HCV strain.
(3) The method according to (2) above, wherein the genotype 1 HCV strain is selected from the group consisting of the H77c strain, the 1 strain, the H strain, the HC-J1 strain, the J1 strain, the con1 strain, the TH strain, the J strain, the JT strain, and the BK strain.
(4) The method according to (2) above, wherein the genotype 2 HCV strain is selected from the group consisting of the J6CF strain, the JFH1 strain, the JCH1 strain, and the HC-J8 strain.
According to the method for producing infectious hepatitis C virus particles from recombinant DNA in a culture cell system of the present invention, infectious HCV particles can be highly produced at a density that is approximately 60 times as high as densities obtained by conventional methods.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.
1. Construction of an HCV Expression Vectors with the Use of an RNA Polymerase I Promoter/Terminator System
There are 3 types of known RNA polymerases that transcribe RNA. RNA polymerase I transcribes rRNA from a ribosomal RNA gene. RNA polymerase II transcribes mRNA from a protein coding gene. RNA polymerase III transcribes tRNA from a tRNA gene.
Each genomic RNA transcribed with RNA polymerases I and III has its own terminator. Transcription is terminated based on the terminator sequence. In contrast, in the case of transcription caused by RNA polymerase II, no terminator sequence is required. The mechanism of the transcription termination is unknown. However, it is considered that what is important for the formation of the 3′ end of mRNA is not transcription termination itself but a cleavage reaction of a primary transcription product.
Unlike the cases of a transcription product of a gene located downstream of an RNA polymerase II promoter, cap and polyA are not added to the 5′ and 3′ ends of each transcription product of genes ligated downstream of RNA polymerase I and III promoters. Natural-occurring HCV genomic RNA does not have cap and polyA added to the 5′ and 3′ ends thereof. Thus, it is expected that RNA identical to HCV viral genomic RNA can be transcribed by expressing HCV genomic DNA with an RNA polymerase I or III promoter.
As an available promoter, any promoter that does not cause the addition of cap and polyA to the 5′ and 3′ ends, respectively, of transcribed RNA is appropriate. Such promoter may be preferably an RNA polymerase I promoter and more preferably an rRNA gene-derived promoter. In addition, such promoter is derived from an animal, preferably from mouse or human. A particularly preferable RNA polymerase I promoter is a human ribosomal RNA (rRNA) gene promoter.
Further, as a terminator sequence, an RNA polymerase I terminator is appropriate, and it is preferably an rRNA gene-derived terminator Such terminator is derived from an animal, preferably from mouse or human. A particularly preferred RNA polymerase I terminator is a mouse ribosomal RNA (rRNA) gene terminator.
An RNA polymerase I promoter/terminator system is used for reconstruction of influenza virus particles (Neumann, G. et al., Virology, 202(1994) pp. 477-479, Neumann, G. & Kawaoka, Y., Virology 287 (2001) pp. 243-250, JP Patent Publication (Kohyo) No. 2003-520573 A). In the method of the present invention, pHH21, an RNA polymerase I promoter/terminator system-based vector (Neumann G. et al., Proc. Natl. Acad. Sci. USA, 96 (1999) pp. 9345-9350), can be used. pHH21 is an expression vector that contains a human RNA polymerase I promoter as a promoter and a mouse RNA polymerase I terminator as a terminator.
A promoter/terminator and HCV genomic cDNA can be ligated without the addition of an excessive nucleotide sequence therebetween by adding a restriction enzyme BsmBI recognition sequence to the 5′ and 3′ ends of HCV genomic cDNA by PCR, digesting it with BsmBI, and inserting the HCV genome into the BsmBI site of pHH21.
In addition, an expression vector used for the method of the present invention can be newly constructed by adequately ligating the above-mentioned promoter, HCV genomic cDNA, and the terminator.
According to phylogenetic analysis with nucleotide sequences of HCV strains, HCVs are classified into the following 6 types: genotype 1a, genotype 1b, genotype 2a, genotype 2b, genotype 3a, and genotype 3b. Each genotype is further classified into some subtypes. In addition, the full-length genomic sequences of some HCV genotypes have been determined (Simmonds, P. et al., Hepatology, 10 (1994) pp. 1321-1324; Choo, Q. L et al., Science, 244 (1989) pp. 359-362; Okamoto, H et al., J. Gen. Virol., 73(1992) pp. 673-679; Mori, S. et al., and Biochem. Biophis. Res. Commun. 183 (1992) pp. 334-342).
In the present invention, specifically, an HCV strain that can be used includes: genotype 1 (including genotypes 1a and 1b) such as H77c (H77 strain consensus sequence: GenBank accession no. AF011751), the 1 strain (GenBank accession no. M62321), the H strain (GenBank accession no. M67463), the HC-J1 strain (GenBank accession no. D10749), the J1 strain (GenBank accession no. D89815), the con1 strain (GenBank accession no. AJ238799), the TH strain (Wakita, T. et al., J. Biol. Chem., (1994) 269, pp. 14205-14210), the J strain (GenBank accession no. D90208), the JT strain (GenBank accession no. D01171), and the BK strain (GenBank accession no. M58335); genotype 2 (including genotypes 2a and 2b) such as the J6CF strain (GenBank accession no. AF177036), the JFH1 strain (also referred to as the JFH-1 strain: GenBank accession no. AB047639), the JCH1 strain (GenBank accession no. AB047640), and the HC-J8 strain (GenBank accession no. D01221). In addition, a list of GenBank accession numbers of other strains has already been reported and thus they are available (Tokita, T. et al., J. Gen. Virol. (1998) 79, pp. 1847-1857; Cristina J. & Colina R. Virolgy Journal, (2006) 3, pp. 1-8).
HCV genomic RNA-derived cDNA of any of the above genotypes can be used for insertion into an RNA polymerase I promoter/terminator-based vector. Further, chimeric cDNA derived from those of any genotypes can also be used. Preferably, cDNA that is derived from a genotype of which HCV genomic RNA can be autonomously replicated when it is introduced into an HCV permissive cell such as Huh7 can be used (Wakita, T., et al. Nat. Med., 11, (2005) pp. 791-796; Lindenbach B D., et al., Science, 309(2005) pp. 623-626). A further preferred cDNA used herein includes a genomic cDNA sequence (GenBank accession no. AB047639, Kato, T. et al., Gastroenterology, 125 (2003) pp. 1808-1817; SEQ ID NO: 27) corresponding to genome RNA of the JFH1 strain of genotype 2a (JP Patent Publication (Kokai) No. 2002-171978 A).
The genome of hepatitis C virus (HCV) is a single-stranded (+) strand RNA comprising approximately 9600 nucleotides. This genomic RNA comprises the 5′ untranslated region (also denoted as 5′ NTR or 5′ UTR), a translated region composed of a structural region and a non-structural region, and the 3′ untranslated region (also denoted as 3′ NTR or 3′ UTR). HCV structural proteins are encoded in the structural region, and a plurality of non-structural proteins are encoded in the non-structural region.
Such HCV structural proteins (Core, E1, E2, and p7) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) are generated by first translation of the translated region into a single continuous polyprotein and then release of proteins with restricted cleavage of the polyprotein by proteases. Among these structural proteins and non-structural proteins (that is, viral proteins of HCV), “Core” is a core protein, and E1 and E2 are envelope proteins. The non-structural proteins are proteins involved in viral self replication, NS2 is known to have metalloprotease activity, and NS3 is known to have serine protease activity (at one-third of the N terminal side) and helicase activity (at two-thirds of the C-terminal side). Furthermore, it has been reported that NS4A is a cofactor for protease activity of NS3, and NS5B has RNA-dependent RNA polymerase activity.
An expression vector used for the method for producing HCV particles of the present invention should comprise: DNA having a sequence containing the 5′ untranslated region, Core protein coding sequence, E1 and E2 protein coding sequences, a p7 protein coding sequence, NS2, NS3, NS4 (including NS4A and NS4B), NS5A, and NS5B protein coding sequences, and the 3′ untranslated region of HCV genomic cDNA in such order, downstream of a promoter recognized by an RNA polymerase I (RNA polymerase I promoter); and a terminator recognized by an RNA polymerase I (RNA polymerase I terminator), further downstream thereof. These sequences are generated by first translation of them into a single continuous polyprotein and then release of proteins with restricted cleavage of the polyprotein by proteases. As a result, HCV particles are produced.
In the method for producing HCV particles of the present invention, an expression vector used herein comprises a DNA fragment in which cDNA synthesized from HCV genomic RNA derived from any HCV strain is ligated downstream of an RNA polymerase I promoter, and an RNA polymerase I terminator is further ligated downstream thereof. HCV cDNA that is to be ligated downstream of an RNA polymerase I promoter and upstream of an RNA polymerase I terminator may be genomic cDNA corresponding to genomic RNA derived from a single HCV strain (preferably the JFH1 strain), or a chimeric nucleic acid that is derived from cDNAs synthesized from genomic RNAs derived from two or more HCV strains (preferably at least one of them is the JFH1 strain). Preferably, the HCV cDNA that is to be ligated between an RNA polymerase I promoter and an RNA polymerase I terminator comprises an HCV entire genome-like cDNA sequence containing DNA sequences encoding the 5′ untranslated region, structural proteins (Core, E1, E2, and p7), non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), and the 3′ untranslated region of HCV in such order.
In particular, a very preferred one because of its high level of infectious HCV production ability is a chimeric HCV expression vector comprising, downstream of an RNA polymerase I promoter, DNA sequences encoding the 5′ untranslated region and structural proteins derived from an HCV strain (preferably of genotype 1 or 2), respectively, optionally DNA sequences encoding HCV strain-derived non-structural proteins, and subsequently DNA sequences encoding HCV JFH1 strain-derived non-structural proteins and the 3′ untranslated region in the order; and a DNA fragment comprising an RNA polymerase I terminator, further downstream thereof.
A more preferred expression vector used in the present invention comprises, downstream of an RNA polymerase I promoter, an HCV genomic cDNA sequence consisting of the 5′ untranslated region coding sequence, a Core protein coding sequence, E1 and E2 protein coding sequences, a p7 protein coding sequence, and an NS2 protein coding sequence of HCV genomic cDNA (i.e., DNA encoding full-length HCV genomic RNA) derived from any HCV strain; and NS3, NS4A, NS4B, NS5A, and NS5B protein coding sequences and the 3′ untranslated region coding sequence of JFH1 strain-derived HCV genomic cDNA; and, further downstream thereof, DNA comprising an RNA polymerase I terminator. In addition, another preferable expression vector comprises: an HCV genomic cDNA sequence comprising, downstream of an RNA polymerase I promoter, the 5′ untranslated region, a Core protein coding sequence, E1 and E2 protein coding sequences, and a p7 protein coding sequence of HCV genomic cDNA derived from any HCV strain; and NS2, NS3, NS4A, NS4B, NS5A, and NS5B protein coding sequences and the 3′ untranslated region coding sequence of JFH1 strain-derived HCV genomic cDNA; and, further downstream thereof, DNA comprising an RNA polymerase I terminator.
Any HCV strain used in the above expression vector can be preferably HCV strains of genotypes 1 and 2. In addition, preferably, the 5′ untranslated region coding sequence in the above expression vector may be a JFH1 strain-derived sequence or a chimeric sequence derived from those of two or more HCV strains selected from genotype 1 and genotype 2 HCV strains. When a chimeric sequence is used, it preferably comprises a JFH1 strain-derived sequence. A genotype-1 HCV strain that can be used herein includes, for example, the H77c strain, the 1 strain, the H strain, the HC-J1 strain, the J1 strain, the con1 strain, the TH strain, the J strain, the JT strain, and the BK strain. A genotype-2 HCV strain that can be used herein includes, for example, the J6CF strain, the JFH1 strain, the JCH1 strain, and the HC-J8 strain. Further preferred HCV strain includes the JFH1 strain, the J6CF strain, the J1 strain, and the H77c strain.
As an example, in JFH1 strain-derived full-length genomic cDNA, a region encoding from the 5′ untranslated region to the NS2 protein, which can be incorporated into the expression vector of the present invention, ranges from nucleotide numbers 1 to 3430 of the nucleotide sequence shown in SEQ ID NO: 27 (DNA sequence with GenBank accession no. AB047639); and a region encoding from the NS3 protein to the 3′ untranslated region ranges from nucleotide numbers 3431 to 9678 thereof. Similarly, in JFH1 strain-derived full-length genomic cDNA, a region encoding from the 5′ untranslated region to the p7 protein ranges from nucleotide numbers 1 to 2779 of the nucleotide sequence shown in SEQ ID NO: 27; and a region encoding from the NS2 protein to the 3′ untranslated region ranges from nucleotide numbers 2780 to 9678 thereof. Also, positions of regions on another HCV strain-derived genomic cDNA can be defined based on the above genomic cDNA sequence of the JFH1 strain.
A preferred example of the expression vector according to the present invention is an expression vector comprising any one of DNA fragments J6(C-p7)JFH1 (SEQ ID NO: 29), H77c(C-p7)JFH1 (SEQ ID NO: 30), J1(C-p7)JFH1 (SEQ ID NO: 31), and J1(C-NS2)JFH1 (SEQ ID NO: 32) shown in the Examples as described below. Preferably, these DNA fragments are inserted under the control of an expression promoter in the vector.
It has been demonstrated that autonomous replication does not take place when amino acid sequence GDD is mutated into GND in the HCV non-structural protein NS5B (Kato, T. et al., Gastroenterology, 125(2003) pp. 1808-1817). Thus, as an experimental control, the NS5B protein having the mutated GND-containing amino acid sequence can be used.
2. Confirmation of Intracellular HCV RNA Synthesis
HCV RNA can be transcribed by introducing the expression vector produced as described above of the present invention into cells. It is possible to introduce DNA into cells by a common method, such as electroporation, a lipofection method, or a calcium phosphate method.
HCV RNA transcribed from expression vector DNA can be analyzed by a conventional molecular biological method (Molecular Cloning 3rd Edition Sambrook & Russell Cold Spring Harbor Laboratory Press 2001). Specifically, it is possible to analyze the amount or the sequence of transcribed RNA by the Northern blot method, a ribonuclease protection assay method, the RT-PCR method, the RACE method, or the like. Upon RNA quantification, the Northern blot method, the RT-PCR method, or the like is used. Upon RNA sequence analysis, the RACE method is used.
When the sequence of the 5′ end of HCV RNA intracellularly transcribed is analyzed, a synthetic RNA linker having a given sequence is ligated to HCV RNA with an RNA ligase and then the resultant is used as a template for cDNA synthesis with a synthetic DNA primer complementary to HCV RNA and a reverse transcription enzyme. Subsequently, PCR is carried out with a primer of a sequence within the linker and a primer located at the 5′ side of the primer used above for amplification of a fragment complementary to HCV RNA. Then the amplification product is cloned into a plasmid vector, followed by nucleotide sequence determination thereof. Accordingly, the sequence of HCV RNA intracellularly transcribed can be analyzed. If a DNA fragment is not amplified in the first PCR, nested-PCR method can be used for performing the sequence analysis.
3. Confirmation of Intracellular HCV Protein Expression
In cells infected with HCV, in which HCV genomic RNA is replicated, the above HCV proteins are expressed. Thus, if HCV proteins are detected in a protein extract from HCV genomic RNA-replicating cells, it can be determined that such cells replicate HCV genomic RNA. Detection of HCV proteins can be carried out in accordance with any known protein detection method. For instance, such detection can be carried out by reacting an antibody against an HCV protein that is expected to be expressed from an introduced HCV genomic RNA with a protein extract from cells. More specifically, for example, such detection can be carried out by blotting a protein sample extracted from cells onto a nitrocellulose membrane, reacting it with an anti-HCV protein antibody (e.g., an anti-NS3-specific antibody or an antiserum collected from a hepatitis C patient), and detecting the anti-HCV protein antibody thereon.
A host cell used for expressing the HCV protein therein may be any cell as long as it can be subcultured. Such a host cell may be preferably a eukaryotic cell, more preferably a human cell, and still more preferably a human liver-derived cell, a human uterine cervix-derived cell, or a human fetal kidney-derived cell. Preferred examples of the cells include proliferative cells such as those of cancer cell lines and stem cell lines. More preferred examples thereof include a Huh7 cell (ATCC CCL-185), a HepG2 cell (ATCC HB 8065), an IMY-N9 cell (Date, T. et al., J. Biol. Chem., (2004) 279, pp. 22371-22376), a HeLa cell (ECACC 93021013), an RCYM1RC cell (Murakami, K., et al., Virology, 351, 381-392, 2006), a 5-15RC cell (Pietschmann T., et al., J Virol. (2001) 75, pp. 1252-1264), and cells of cell lines derived from such cells. Particularly preferred cells include a Huh7 cell, an RCYM1RC cell, a 5-15RC cell, a HepG2 cell, and cells of cell lines derived from such cells. These cells used herein may be commercially available cells, or may be obtained from cell depositories, or may be cells of cell lines established from any cells (e.g., cancer cells or stem cells). Cells of cell lines derived from a Huh7 cell include a Huh7.5 cell (Blight, K J. Et al., J. Virol., (2002) 76, pp. 13001-13014) and a Huh7.5.1 cell (Zhong, J. et al., Proc. Natl. Acad. Sci USA, (2005) 102, pp. 9294-9299). The former is a cell with high HCV replication ability, which is obtained by eliminating a replicon with an interferon treatment from a replicon-replicating cell that has been established through gene introduction of a replicon into a Huh7 cell. The latter is a cell with good replicon replication efficiency, which is obtained by eliminating a replicon with an interferon γ treatment from a replicon-replicating cell generated from a Huh7.5 cell.
4. Confirmation of HCV Particle Production
The hepatitis C virus (HCV) particle comprising HCV genomic RNA transcribed from the expression vector of the present invention can be produced by introducing the expression vector of the present invention into an HCV permissive cell (cell that allows HCV proliferation) and culturing the cell resulting that. Preferred HCV permissive cell used herein includes a Huh7 cell, an RCYM1RC cell, a 5-15RC cell, a HepG2 cell, and cells of cell lines derived from such cells. The thus produced hepatitis C virus (HCV) particle has the ability to infect other cells. The present invention relates to such a method for producing the infectious hepatitis C virus particle.
The virus particle production ability of the cells may be confirmed by any known virus detection method. For instance, the culture solution of cells that are suspected of producing virus particles is fractionated through the sucrose density gradient, and the density of fraction, HCV core protein concentration, and amount of HCV genomic RNA are determined for each fraction. As a result, if the peak of the HCV core protein coincides with that of HCV genomic RNA, and if the density of the fraction in which the peak was detected is smaller than the density of the equivalent fraction as obtained by fractionating the culture supernatant treated with 0.25% NP40 (Polyoxyethylene(9)Octylphenyl Ether), the cells used can be determined to have virus particle production ability. Alternatively, since it has been reported that free HCV particles have specific gravity values of 1.14 to 1.16 g/ml (Kaito, M. et al., J. Gen. Virol. 75 (1994) pp. 1755-1760), it is also possible to compare the above values with the specific gravity.
Alternatively, HCV virus particles released into a culture solution can be detected with an antibody against the Core protein, E1 protein, E2 protein, or the like. In addition, the presence of HCV virus particles can be indirectly detected by amplifying HCV genomic RNA contained in HCV virus particles in a culture solution by the RT-PCR method with specific primers, followed by detection.
5. Infection of Another Cell with HCV Particles of the Present Invention
HCV virus particles produced by the method of the present invention have the ability to infect a cell (preferably an HCV permissive (sensitive) cell). According to the present invention, a method for producing a hepatitis C virus-infected cell comprising culturing a cell into which an HCV expression vector based on an RNA polymerase I promoter/terminator system has been introduced and infecting another cell (preferably an HCV permissive cell) with virus particles in the obtained culture (preferably culture supernatant), is also provided. In the present invention, the HCV permissive cell means a cell having an ability to replicate HCV genomic RNA and/or to be infected with HCV. Such HCV permissive cell is preferably, but is not limited to, a hepatic cell or a lymphoid lineage cell. Specifically, the hepatic cell includes, but is not limited to, a primary hepatocyte, a Huh7 cell, a HepG2 cell, an IMY-N9 cell, a HeLa cell, a 293 cell, and the like. The lymphoid lineage cell includes, but is not limited to, a Molt4 cell, an HPB-Ma cell, a Daudi cell, and the like. Particularly preferred examples of such HCV permissive cell include a Huh7 cell, an RCYM1RC cell, a 5-15RC cell, a HepG2 cell, and cells of cell lines derived (produced) from such cells. Preferred examples of a cell derived from a Huh7 cell include a Huh7.5 cell and a Huh7.5.1 cell.
When a cell (e.g., an HCV permissive cell) is infected with HCV particles generated in a cell into which the HCV expression vector based on an RNA polymerase I promoter/terminator system of the present invention has been introduced, HCV genomic RNA is replicated and virus particles are formed in the infected cell.
HCV virus particles generated in the cell into which the HCV expression vector based on an RNA polymerase I promotor/terminator system of the present invention has been introduced can infect HCV-susceptible animals such as chimpanzees and the like and induce hepatitis caused by HCV therein.
It is possible to determine whether or not HCV particles generated in a cell into which the HCV expression vector based on an RNA polymerase I promotor/terminator system of the present invention has been introduced have infectivity in the following manner. An HCV permissive cell (e.g., Huh7) is treated with a culture supernatant obtained by culturing a cell into which the HCV expression vector comprising an RNA polymerase I promotor/terminator system of the present invention has been introduced. 48 hours later, for instance, the cells are immunostained with an anti-core antibody and then infected cells are counted. Alternatively, a cell extract is subjected to SDS-polyacrylamide gel electrophoresis and then core protein can be detected by Western blotting.
6. Obtaining Infectious HCV Particle-Producing Cell Lines
A cell into which the HCV expression vector based on an RNA polymerase I promotor/terminator system of the present invention has been stably introduced can continuously produce infectious HCV particles. In order to obtain a cell line in which the HCV expression vector based on an RNA polymerase I promotor/terminator system of the present invention is stably expressed, it is preferable to incorporate a drug-resistant gene into the vector. Examples of a drug-resistant gene include a G418-resistant gene, a hygromycin-resistant gene, a puromycin-resistant gene, a Zeocin-resistant gene, and a blasticidin-resistant gene. In addition, it is possible to estimate the HCV particle production ability of a clone selected based on drug resistance by detecting the amount of Core protein in a culture supernatant of such clone or the amount of HCV RNA replicated in such clone by Northern blotting or quantitative RT-PCR.
This description includes the disclosure of the description and/or drawings of Japanese Patent Application No. 2005-287646, to which the present application claims a priority.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
The present invention is hereafter described in greater detail with reference to the following examples. However, the technical scope of the present invention is not limited thereto.
Construction of HCV Expression Vectors with the Use of the RNA Polymerase I Promotor/Terminator System
cDNA of HCV genomic RNA used were cDNA of JFH1 strain-derived genomic RNA of genotype 2a (GenBank accession no. AB047639, Kato, T. et al., Gastroenterology, 125(2003) pp. 1808-1817), cDNA of JFH1 strain-derived genomic RNA having a DNA sequence modified to cause a GDD amino acid sequence in NS5B of the JFH1 strain to be replaced by a GND amino acid sequence (Wakita, T. et al. Nat. Med., 11 (2005) pp. 791-796), cDNA of J6CF strain-derived genomic RNA (GenBank accession no. AF177036, Yanagi, M. et al. Virology, 262 (1999) pp. 250-263), cDNA of H77c strain-derived genomic RNA of genotype 1a (GenBank accession no. AF011751, Yanagi, M. et al., Proc. Natl. Acad. Sci. USA, 94 (1997) pp. 8738-8743), and cDNA of J1 strain-derived genomic RNA of genotype 1b (GenBank accession no. D89815, Aizaki, H. et al. Hepatology, 27 (1998) pp. 621-627).
A restriction enzyme BsmBI recognition sequence was added to the 5′ end and the 3′ end of each of the above HCV genomic cDNAs (JFH1, JFH1/GND, and H77c) by PCR. A BsmBI cleavage site is located at a distance from a BsmBI recognition site. With the utilization of such property, each HCV genome was inserted into the BsmBI site of a pHH21 vector having the human RNA polymerase I promoter sequence and the mouse RNA polymerase I terminator sequence (Neumann G. et al., Proc. Natl. Acad. Sci. USA, 96 (1999) pp. 9345-9350) without insertion of extra nucleotide sequences between the promoter/terminator and the HCV genome (
Further, chimeric HCV expression vectors derived from genomic cDNAs of J6CF and JFH1, those of H77c and JFH1, and those of J1 and JFH1 were produced in the manner described below.
Production of pHH J6(C-p7)/JFH1
A plasmid DNA pJFH1 constructed by cloning cDNA corresponding to the JFH1 strain-derived entire genomic RNA region into a pUC19 plasmid (Wakita, T. et al. Nat. Med., 11 (2005) pp. 791-796, WO 2004/104198) was digested with AgeI, followed by further partial digestion with BclI. And then, the resulting plasmid DNA fragment from which a fragment ranging from the AgeI site to the first BclI site (2672 bp) had been removed was purified. Meanwhile, a 2672-bp fragment obtained by partial digestion of J6CF-strain-derived genomic cDNA with AgeI and BclI was ligated to the above purified fragment to obtain pUC J6/JFH1.
Next, an approximately 4.3 kb of fragment obtained by digestion of pHH JFH1 with NocI and an approximately 8.2 kb of fragment obtained by digesting of pUC J6/JFH1 with NocI were ligated with a ligase to obtain an expression vector pHH J6(C-p7)/JFH1. The insert (J6(C-p7)JFH1; SEQ ID NO: 29,
Production of pHH H77c(C-p7)/JFH1
To the above JFH1 genomic cDNA as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 5-JFH-S (SEQ ID NO: 10: GCCATGGCGTTAGTATGAGTGTCGT) and 5-JFH-A (SEQ ID NO: 11: TCGTGCTCATGGTGCACGGTCTACGAGACC) (1 μl each) were added and then deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 30 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 1. Then, to the JFH1 genomic cDNA as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 3-JFH-S (SEQ ID NO: 12: GGCATACGCATATGACGCACCTGTGCACGG) and 3-JFH-A (SEQ ID NO: 13: GCTCTGACGAAGTACGGCACATGTGTC) (1 μl each) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 2. Further, to the above H77c genomic cDNA as a template, 10× buffer (5 μl), and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 5-H77-S (SEQ ID NO: 14: ACCGTGCACCATGAGCACGAATCCTAAACC) and 5-H77-A (SEQ ID NO: 15: GAAGCCGCACGTAAGGGTATCGATG) (1 μl each) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 3. Then, to the H77c genomic cDNA as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 3-H77-S (SEQ ID NO: 16: CATTGTGCCCGCAAAGAGCGTGTGT) and 3-H77-A (SEQ ID NO: 17: GTGCGTCATATGCGTATGCCCGCTGAGGCA) (1 μl each) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 4.
Next, each PCR product was purified and dissolved in 50 μl of H2O. DNAs of PCR products nos. 1 and 3 were separately 100-fold diluted and 1 μl of each were mixed together. The resulting mixture was used as a template and PCR was carried out for 5 cycles under the above conditions without the addition of primers. Thereafter, primers 5-JFH-S (SEQ ID NO: 10) and 5-H77-A (SEQ ID NO: 15) were added thereto, followed by PCR for 25 cycles. The thus amplified chimeric DNA fragment was purified. The fragment was cloned into a plasmid vector pCRII. The proper nucleotide sequence was verified by sequencing the DNA fragment. This plasmid obtained by cloning the chimeric DNA fragment into pCRII was designated as pCR5HJ. Further, PCR products nos. 2 and 4 were separately 100-fold diluted and 1 μl of each were mixed together. The resulting mixture was used as a template and PCR was carried out for 5 cycles under the above conditions without the addition of primers. Thereafter, primers 3-H77-S (SEQ ID NO: 16) and 3-JFH-A (SEQ ID NO: 13) were added thereto, followed by PCR for 25 cycles. The thus amplified chimeric DNA fragment was purified. The fragment was cloned into a plasmid vector pCRII. The proper nucleotide sequence was verified by sequencing the DNA fragment. This plasmid obtained by cloning the chimeric DNA fragment into pCRII was designated as pCR3HJ.
Next, pCR5HJ was digested with restriction enzymes AgeI and KpnI, H77c genomic cDNA was digested with restriction enzymes KpnI and AscI, and pCR3HJ was digested with restriction enzymes AscI and NotI. Each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. The three DNA fragments above were ligated to a vector obtained by digesting pHH JFH1 with AgeI and NotI. The vector was designated as pHH H77c(C-p7)/JFH. The insert (H77c(C-p7)JFH; SEQ ID NO: 30,
Production of pHH J1(C-p7)/JFH1
To the above JFH1 genomic cDNA as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 5-JFH-S (SEQ ID NO: 10: GCCATGGCGTTAGTATGAGTGTCGT) and 5-JFH-A2 (SEQ ID NO: 18: TTGTGCTCATGGTGCACGGTCTACGAGACC) (1 μl each) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 5.
Next, to the JFH1 genomic cDNA as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 3-JFH-S2 (SEQ ID NO: 19: AGCTTACGCCTATGACGCACCTGTGCACGG) and 3-JFH-A (SEQ ID NO: 13: GCTCTGACGAAGTACGGCACATGTGTC) (1 μl each) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 6.
To cDNA corresponding to the genomic RNA derived from the J1 strain of genotype 1b (GenBank accession no. D89815, Aizaki, H. et al. Hepatology, 27 (1998) pp. 621-627) as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 5-J1-S (SEQ ID NO: 20: ACCGTGCACCATGAGCACAAATCCTAAACC) and 5-J1-A (SEQ ID NO: 21: AAGCGGGATGTACCCCATGAG) (each 1 μl) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 7.
Next, to the above J1 strain-derived genomic cDNA as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 3-J1-S (SEQ ID NO: 22: CGGCTGTACATGGATGAATAGCACTGGGTT) and 3-J1-A (SEQ ID NO: 23: GTGCGTCATAGGCGTAAGCTCGTGGTGGTA) (1 μl each) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 8.
Next, each PCR product was purified and dissolved in 50 μl of H2O. DNAs of PCR products nos. 5 and 7 were separately 100-fold diluted and 1 μl of each were mixed together. The resulting mixture was used as a template and PCR was carried out for 5 cycles under the above conditions without the addition of primers. Thereafter, primers 5-JFH-S (SEQ ID NO: 10) and 5-J1-A (SEQ ID NO: 21) were added thereto, followed by PCR for 25 cycles. The thus amplified chimeric DNA fragment was purified. The fragment was cloned into a plasmid vector pCRII. The nucleotide sequence was verified by sequencing the DNA fragment. The plasmid obtained by cloning the chimeric DNA fragment into pCRII was designated as pCR5JJ.
Further, DNAs of PCR products nos. 6 and 8 were separately 100-fold diluted and 1 μl of each were mixed together. The resulting mixture was used as a template and PCR was carried out for 5 cycles under the above conditions without the addition of primers. Thereafter, primers 3-J1-S (SEQ ID NO: 22) and 3-JFH-A (SEQ ID NO: 13) were added thereto, followed by PCR for 25 cycles. The thus amplified chimeric DNA fragment was purified. The fragment was cloned into a plasmid vector pCRII. The proper nucleotide sequence was verified by sequencing the DNA fragment. The plasmid obtained by cloning the chimeric DNA fragment into pCRII was designated as pCR3JJ.
pCR5JJ was digested with restriction enzymes AgeI and ClaI, J1 genomic cDNA was digested with restriction enzymes ClaI and AvrII, and pCR3JJ was digested with restriction enzymes AvrII and KpnI. Each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. The above three DNA fragments were ligated to a vector obtained by digesting pHH JFH1 with AgeI and KpnI. The vector was designated as pHH J1(C-p7)/JFH. The insert (J1(C-p7)JFH; SEQ ID NO: 31,
Production of pHH J1(C-NS2)/JFH1
To the above JFH1 genomic cDNA as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 5-JFH-S (SEQ ID NO: 10: GCCATGGCGTTAGTATGAGTGTCGT) and 5-JFH-A2 (SEQ ID NO: 18: TTGTGCTCATGGTGCACGGTCTACGAGACC) (1 μl each) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 9.
Next, to the JFH1 genomic cDNA as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 3-JFH-NS3-S (SEQ ID NO: 24: GCGACTCCTTGCTCCCATCACTGCTTATGC) and 3-JFH-NS3-A (SEQ ID NO: 25: TGGGAGACCTTGTAACAACGTCGAGTGT) (1 μl each) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 10.
To cDNA of the genomic RNA derived from the J1 strain of genotype 1b (GenBank accession no. D89815, Aizaki, H. et al. Hepatology, 27 (1998) pp. 621-627) as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 5-J1-S (SEQ ID NO: 20: ACCGTGCACCATGAGCACAAATCCTAAACC) and 5-J1-A (SEQ ID NO: 21: AAGCGGGATGTACCCCATGAG) (1 μl each) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 11.
Next, to J1 strain-derived genomic cDNA as a template, 10× buffer (5 μl) and 2.5 mM dNTP mixture (5 μl) which were attached to an LA-PCR kit (Takara Bio Inc.), and 10 μM primers 3-J1-S (SEQ ID NO: 22: CGGCTGTACATGGATGAATAGCACTGGGTT) and 3-J1-NS3-A (SEQ ID NO: 26: CATAAGCAGTGATGGGAGCAAGGAGTCGCC) (1 μl each) were added, and then, deionized water was added up to a final total volume of 49 μl. Subsequently, Takara LA Taq (Takara Bio Inc.) (1 μl) was added thereto, followed by a PCR reaction. The PCR reaction was carried out under conditions of 25 cycles of 94° C. for 1 minute, 64° C. for 2 minutes, and 72° C. for 3 minutes. The thus obtained PCR product was designated as a PCR product no. 12.
Each PCR product was purified and dissolved in 50 μl of H2O. DNAs of PCR products nos. 9 and 11 were separately 100-fold diluted and 1 μl of each were mixed together. The resulting mixture was used as a template and PCR was carried out for 5 cycles under the above conditions without the addition of primers. Thereafter, primers 5-JFH-S (SEQ ID NO: 10) and 5-J1-A (SEQ ID NO: 21) were added thereto, followed by PCR for 25 cycles. The thus amplified chimeric DNA fragment was purified. The fragment was cloned into a plasmid vector pCRII. The proper nucleotide sequence was verified by sequencing the DNA fragment. The plasmid obtained by cloning the chimeric DNA fragment into pCRII was designated as pCR5JJ.
Further, DNAs of PCR products nos. 10 and 12 were separately 100-fold diluted and 1 μl of each were mixed together. The resulting mixture was used as a template and PCR was carried out for 5 cycles under the above conditions without the addition of primers. Thereafter, primers 3-J1-S (SEQ ID NO: 22) and 3-JFH-NS3-A (SEQ ID NO: 25) were added thereto, followed by PCR for 25 cycles. The thus amplified chimeric DNA fragment was purified. The fragment was cloned into a plasmid vector pCRII. The proper nucleotide sequence was verified by sequencing the DNA fragment. The plasmid obtained by cloning the chimeric DNA fragment into pCRII was designated as pCR3JJNS3.
pCR5JJ was digested with restriction enzymes AgeI and ClaI, J1 genomic cDNA was digested with restriction enzymes ClaI and AvrII, and pCR3JJNS3 was digested with restriction enzymes AvrII and BspDI. Each HCV cDNA fragment was fractionated by agarose gel electrophoresis and purified. The three DNA fragments above were ligated to a vector obtained by digesting pHH JFH1 with AgeI and BspDI. The vector was designated as pHH J1(C-NS2)/JFH. The insert (J1(C-NS2)JFH; SEQ ID NO: 32,
Confirmation of Intracellular HCV RNA Synthesis
pHH JFH1 and pHH JFH1/GND produced in Example 1 were introduced into Huh7 and HepG2 cells using Fugene 6 (Roche) in accordance with the attached manufacturer's instructions. 24 hours later, RNA was prepared from each cell with Isogen (Nippon Gene Co., Ltd.) in accordance with the attached manufacturer's instructions.
As described below, it was confirmed whether or not HCV RNA was synthesized from pHH JFH1 and pHH JFH1/GND by the RACE method using the obtained RNAs as templates.
2 μg each RNA prepared above were separately ligated to 2.5 μM RNA linker (SEQ ID NO: 1: GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA) with T4 RNA ligase (Takara) and an attached buffer. Then, cDNA synthesis was carried out with RNA linker-ligated RNA as a template, a primer (SEQ ID NO: 2: gtaccccatgaggtcggcaaag) complementary to HCV RNA, and the SuperScript III reverse-transcription enzyme (Invitrogen Corporation) in accordance with the attached manufacturer's instructions.
Next, DNA amplification was carried out by PCR using each synthesized cDNA described above as a template and two types of primers, which were a 5′ end sense primer (SEQ ID NO: 3: GCTGATGGCGATGAATGAACACTG) for RNA linker and a 3′ end antisense primer (SEQ ID NO: 4:gaccgctccgaagttttccttg). Further, a second PCR step was carried out with each amplified DNA as a template and a primer set of a 5′ end primer (SEQ ID NO: 5:GAACACTGCGTTTGCTGGCTTTGATG) and a 3′ end primer (SEQ ID NO: 6: cgccctatcaggcagtaccacaag) corresponding to the inside sequence of the amplified DNA. This PCR was carried out with a commercially available kit, Ex Taq kit (Takara) as follows: heating treatment at 96° C. for 5 minutes; 35 cycles of 96° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes; and preservation at 4° C. Subsequently, in order to confirm the production of the 2nd PCR product, a portion of the reaction solution was subjected to agarose gel electrophoresis for confirmation. As a result (
Confirmation of Intercellular HCV Protein Expression
It was confirmed whether or not HCV protein translation took place following transcription in cells into which pHH JFH1, pHH H77c, and pHH JFH1/GND had been separately introduced, as described below.
pHH JFH1, pHH H77c, and pHH JFH1/GND were separately introduced into Huh7 cells using Fugene 6 (Roche) in accordance with the attached manufacturer's instructions. On the 4th day after culture, cell extracts were prepared by a conventional method. Then, proteins in the extracts were analyzed by SDS-PAGE and the Western blot method. Upon analysis, a cell extract obtained by transiently transfecting expression plasmid DNA containing the Core gene into a Huh7 cell was used as a positive control. Further, a cell extract obtained from an untransfected Huh7 cell was used as a negative control. A sample extracted from each cell clone was subjected to SDS-PAGE, followed by blotting on a PVDF membrane (Immobilon-P, Millipore). The Core protein and the NS5A protein translated in each cell that had been blotted on the membrane were detected using ECL (Amersham Pharmacia) with an anti-Core-specific antibody (rabbit polyclonal antibody), a mouse monoclonal antibody (Austral) as an anti-NS5A antibody, and HRP-labeled secondary antibodies capable of recognizing the above antibodies.
Consequently, as shown in
Next, examination took place to identify the cell lines in which HCV protein expression could be caused. pHH JFH1 and pHH JFH1/GND were introduced together with a GFP expression vector into Huh7, RCYM1RC, 5-15RC, HepG2, and 293T cells (pGreenLantern: Life Technologies Inc.) with Fugen 6 (Roche) in accordance with the attached manufacturer's instructions. On the 4th day after introduction, GFP expression was observed with a fluorescence microscope so that introduction of each vector was confirmed. Then, a cell extract was prepared from each cell. Subsequently, the Core protein in each extract was analyzed by SDS-PAGE and the Western blot method. The Core protein was detected using ECL (Amersham Pharmacia) with an anti-Core-specific rabbit polyclonal antibody and an HRP-labeled secondary antibody capable of recognizing the antibody. As a result, as shown in
Production of HCV Particles
Next, in order to confirm whether or not HCV particles would be produced by a cell into which pHH JFH1 had been introduced, the Core protein in a culture supernatant was analyzed. pHH JFH1 and, as a control, a vector pCAG327JFH1 expressing Core protein, E1, E2 and p7 were separately introduced into HepG2 cells using Fugene 6. On the 2nd day after introduction, each culture supernatant was removed, followed by the addition of a fresh medium and further culturing for 2 days. Thereafter, cells and culture solutions were collected, followed by protein extraction from the cells. The Core protein expression in each cell was analyzed by the method shown in Example 3. As a result, the Core protein expression was confirmed (
As shown in
If Core protein and HCV RNA found in fractions at 1.15 to 1.18 mg/ml form an HCV particle structure, such particles would be sensitive to surfactant NP40 treatment. Thus, a culture solution obtained 2 to 4 days after introduction of pHH JFH1 into HepG2 was treated with 0.2% NP40 for 20 minutes, followed by fractionation through a sucrose density gradient. As shown in
Based on the above results, it was confirmed that introduction of pHH JFH1 into a HepG2 cell resulted in transcription of viral RNA from the HCV genomic cDNA, followed by viral protein synthesis and viral RNA replication, and thus virus particles were formed and secreted in a culture solution.
Confirmation of Infectivity of HCV Particles
It was examined whether or not HCV particles obtained in Example 4 would be infectious. pHH JFH1 was introduced into each of Huh7 and HepG2 cells using Fugene 6. Culture supernatants cultured for 3 to 5 days were 30-fold concentrated with an ultrafilter membrane (cut off: 1×105 Da). Then, Huh7.5.1 cells were cultured on 15-mm coverslips in the concentrated culture solutions (100 μl each) containing HCV particles. On the 4th day, the cells were fluorescent-stained with an anti-NS5A antibody. Anti-NS5A antibody-staining-positive cells (namely, infected cells) were counted. As a result, as shown in
Obtaining an Infectious HCV Particle-Producing Cell Line
With the use of the system shown in the above Examples, it was attempted to establish a cell line that continuously produces infectious HCV particles.
A vector was produced by digesting pHH JFH1 with NheI and incorporating into the digested site an expression unit (SV40 promotor/Zeo/polyA) obtained by ligating the Zeocin-resistant gene downstream of an SV40 promoter that had been obtained by digesting pSV40/Zeo2 (Invitrogen) with NheI and XbaI and ligating an SV40 polyA addition signal further downstream thereof. The obtained vector was designated as pHH/ZeoJFH1 (
pHH/ZeoJFH1 was introduced into HepG2 cells using Fugene 6, followed by culture in a Zeocin-containing medium. Then, culture in a Zeocin-containing medium was continued, with a culture solution being exchanged twice a week. The viable cell colony found in a culture dish prepared on the 21st day of culture was cloned. Then, culture was continued. As a result of such colony cloning, 100 cell clones were obtained. The Core protein amount in a culture supernatant of such a clone was determined by the Ortho HCV antigen IRMA test (Aoyagi et al., J. Clin. Microbiol., 37 (1999) p. 1802-1808). A HepG2/No59 cell clone having a high Core protein expression level was selected.
HepG2/No59 cells (2×106 cells) were cultured in a medium (8 ml) with a 10-cm dish. 48 hours later, the culture supernatant was collected.
As a result of assay of the Core protein in the culture supernatant, the HepG2/No59 cell was found to produce 1,400 fmol/L of Core protein.
In addition, RNA prepared from the culture supernatant was subjected to measurement of HCV genomic RNA amount. HCV RNA was detected by quantitative RT-PCR according to the method of Takeuchi et al. (Takeuchi T. et al., Gastroenterology, 116 (1999) pp. 636-642), during which RNA in the 5′ untranslated region of HCV RNA was detected. Specifically, HCV RNA contained in RNA extracted from cells was subjected to PCR amplification with the following synthesis primers and an EZ rTth RNA PCR kit (Applied Biosystems), followed by detection with an ABI Prism 7700 sequence detector system (Applied Biosystems).
The results showed that there were 6.1×108 copies/ml of HCV RNA in the HepG2/No59 cell culture supernatant. Such production amount was approximately 60 times as large as those reported in the past.
Further, the HepG2/No59 cell culture supernatant was 30-fold concentrated with an ultrafilter membrane (cut off: 1×105 Da). Then, Huh7.5.1 cells were cultured on 15-mm coverslips in the concentrated culture solution (100 μl) containing HCV particles. On the 4th day, the cells were immunostained with an anti-NS5A antibody. As a result, Anti-NS5A antibody-staining-positive cells, namely infected cells, were detected. Accordingly, HCV particles secreted in a HepG2/No59 cell culture solution were confirmed to exhibit infectivity.
Production of HCV Particles with Chimeric HCV Expression Vectors
Chimeric HCV expression vectors pHH H77c(C-p7)/JFH1, pHH J6(C-p7)/JFH1, pHH J1(C-p7)/JFH1, and pHH J1(C-NS2)/JFH1 produced in Example 1 were introduced into Huh7.5.1 cells, which were of an established cell line derived from a Huh7 cell (Zhong, J. et al., Proc. Natl. Acad. Sci USA, 102, 9294-9299, (2005)) using Fugene 6 (Roche) in accordance with the attached manufacturer's instructions. On the 3rd day after introduction, the culture supernatants were collected, followed by measurement of the Core protein amount in the culture supernatants. The Core protein amount was measured by the Ortho HCV antigen IRMA test (Aoyagi et al., J. Clin. Microbiol., 37 (1999) pp. 1802-1808). A culture supernatant of a cell into which no vectors had been introduced was used as a negative control. A culture supernatant of a cell into which pHH JFH1 had been introduced was used as a positive control. Table 1 shows examples of the above experimental results. The Core protein was observed in each cell supernatant of cells into which a different chimeric HCV expression vector had been introduced, and therefore it was determined that virus particles were produced. In addition, pHH J6(C-p7)/JFH1 was found to have HCV production ability at a level at least 10 times higher than that of pHH JFH1.
pHH J6(C-p7)/JFH1 and pHH J1(C-p7)/JFH1 were introduced into Huh7.5.1 cells in the manner described in Example 7. The culture supernatants obtained on the 3rd day after introduction were concentrated with an ultrafilter membrane (cut off: 1×105 Da). Then, each the concentrated culture supernatant (100 μl) containing HCV particles was added to Huh7.5.1 cells, followed by culture on 15-mm coverslips. On the 4th day, the cells were immunostained with an anti-NS5A antibody. As a result, cells strongly stained with the anti-NS5A antibody were found among cells treated with the culture supernatant obtained from the cells into which pHH J6(C-p7)/JFH1 had been introduced. In contrast, cells treated with the culture supernatant obtained from the cells into which pHH J1(C-p7)/JFH1 had been introduced were clearly stained when compared with control cells, although the intensity of staining with an anti-NS5A antibody in the case of pHH J1(C-p7)/JFH1 was weaker than that in the case of pHH J6(C-p7)/JFH1. Accordingly, it was revealed that infectious HCV was produced in cells into which pHH J6(C-p7)/JFH1 and pHH J1(C-p7)/JFH1 had been introduced.
In addition, pHH/Zeo J1(C-p7)/JFH1 was produced by inserting a Zeocin-resistant gene expression unit into pHH J1(C-p7)/JFH1 in the manner described in Example 6. Then, pHH/Zeo J1(C-p7)/JFH1 was introduced into Huh7.5.1 such that cells capable of stably expressing virus particles and of propagating in a Zeocin-containing medium were obtained. The culture supernatant (8 ml) of such cells was concentrated with an ultrafilter membrane (cut off: 1×105 Da). The HCV core protein amount in the concentrated culture supernatant was 2365 fmol/L. Huh7.5.1 was infected with the concentrated culture supernatant (100 μl). On the 4th day, cells were immunostained with an anti-NS5A antibody. As a result, cells strongly stained with anti-NS5A antibody were found among cells treated with the culture supernatant obtained from stable expression cells into which pHH/Zeo J1(C-p7)/JFH1 had been introduced. Accordingly, it was revealed that infectious HCV was produced from the above cells.
It was shown that a chimeric HCV expression vector as described above can produce infectious HCV in cells infected therewith.
Free Text of Sequence Listing
The sequence of SEQ ID NO: 1 refers to synthetic RNA.
The sequences of SEQ ID NO: 2 to SEQ ID NO: 9 refer to synthetic DNAs.
The sequences of SEQ ID NOS: 10 to 26 refer to primers.
The sequences of SEQ ID NOS: 29 to 32 refer to chimeric DNAs.
The sequences of SEQ ID NOS: 33 to 45 refer to synthetic DNAs.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-287646 | Sep 2005 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2006/319572 | 9/29/2006 | WO | 00 | 3/28/2008 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2007/037428 | 4/5/2007 | WO | A |
| Number | Date | Country |
|---|---|---|
| 2001-17187 | Jan 2001 | JP |
| WO 2004104198 | Dec 2004 | WO |
| WO-2005080575 | Sep 2005 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20100035345 A1 | Feb 2010 | US |
